The Ras family of small GTPases is composed of intracellular signaling molecules which are regulated by the nucleotide guanosine-5′-triphosphate (GTP). These proteins function as binary signaling switches with both “on” and “off” states. In the “off” state, Ras is bound to the nucleotide guanosine diphosphate (GDP), while in the “on” state, Ras is bound to GTP. Hence, activation and deactivation of Ras are controlled by cycling between the active GTP-bound and inactive GDP-bound forms. In the GTP-bound conformation, Ras has high affinity for numerous effectors which allow it to carry out its intracellular functions. Ras-regulated signaling pathways control processes as diverse as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration.
Mutations in the Ras proto-oncogene family are the most common oncogene-related mutations in human cancer and in tumors in animal models of cancer. Several different mutations in Ras oncogenes are associated with tumor cell phenotypes in nature. For example, mutations at the codon encoding amino acid 12 in the Ras protein are found in 78% of pancreatic cancers, 34% of colorectal cancers, 40% of non-small cell lung adenocarcinomas, and 24% of ovarian cancers. Ras mutations at amino acids 13, 59 and 61 are also found in a variety of other cancers (e.g. see Lu et al., Cancer Res. 2004, 64(15):5084-8; Abrams et al, Sent Oncol, 118-134; and Friday & Adjei, Biochim Biophys Acta, 2005, 1756:127144). These well-characterized mutations at codons 12, 13 and 61 result in constitutive Ras activation (i.e. a Ras protein with the inability to adopt its “off” state, irrespective of whether it is bound to GDP). Consequently, aberrant signaling through the Ras oncogene product pathway plays an important role in uncontrolled cell proliferation and tumorigenesis.
Even though the first two Ras genes were identified in 1982 (Chang et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79 (16): 4848-52) and the major role played by mutant forms of Ras in human cancer has been appreciated for some time, there is currently no single approved therapy which directly targets this most frequently activated of genes in human cancers. Throughout the field of cancer biology, Ras is widely known as an “undruggable” target, due to Ras-effector protein—protein interactions occurring intracellularly within tumor cells, GTP's role in several significant cellular processes unrelated to Ras, the picomolar affinity of GTP for Ras, as well as the critical role of protein-protein interactions between Ras and its accessory proteins in its signaling cascade. (see, e.g., Tanaka & Rabbitts, 2010, Oncogene, 29:6064-6070; Verdine & Walensky, 2007, Clinical Cancer Research, 13:7264). What is needed, therefore, are techniques to identify compounds capable of specifically modulating the behavior of Ras in cancer cells to decrease the aberrant signaling of downstream Ras effector molecules.
Such techniques are provided herein by the disclosure of methods for identifying agents capable of modulating the structure of proteins (such as Ras proteins) into alternate conformational states.
Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.